The present invention relates to a novel bioactive substance which is effective in the prevention or treatment of various diseases accompanied by abnormal angiogenesis.
Angiogenesis is a biological phenomenon which is essential for the formation of a vascular arbor during fetal period and is also essential in the morphological and functional development of each organ. Newly blood vessels are constructed by way of plural processes such as the migration and proliferation of endothelial cells and tube formation. It is reported that in these processes, the participation of mast cells, lymph corpuscles, interstitial cells and others play an important role (J. Folkman and Y. Shing, J. Biol. Chem., 267, 10931, 1992). In adult, the angiogenesis occurs only in the female menstrual cycle and a morbid increase of angiogenesis is involved in various diseases and their progression. Given as specific examples of diseases accompanied by abnormal angiogenesis are cancers, rheumatic arthritis, atherosclerosis, diabetic retinopathy, hemangioma and psoriasis (J. Folkman, N. Engl. J. Med., 333, 1757, 1995).
It is reported that the growth of, particularly, solid cancers depends upon the angiogenesis and an antiangiogenic agent is expected to be a new chemotherapy for intractable solid cancers (Folkman J., J. Natl. Cancer Inst., 82, 4, 1990).
In the case of the rheumatic arthritis, it is reported that over production of angiogenesis factors from infiltrated macrophage/lymphatic cells leads to the induction of angiogenesis in the segments (Kahn J., Proc. Am. Soc. Clin. Oncol., 12, 50, abstract, 1993) and that an antiangiogenic agent AGM-470 inhibits arthritis in a collagen arthritis model (Peacock D J., J. Exp. Med. 175, 1135-8, 1992). These reports suggest that the inhibition of angiogenesis in inflammatory diseases results in the therapy for the diseases.
In the atherosclerosis, it is clarified that excess production of a vascular endothelial growth factor (VEGF) induced at hypoxia causes angiogenesis in an atheromatous macula (Stavri G T., FEBS Lett., 358 , 311-5, 1995) and it is said that angiogenesis is necessary for the extension of the atheromatous macula like in the case where angiogenesis is necessary for the growth of tumors (O""Brien E R., Am. J. Pathol., 146, 1029-39, 1994). For hemangioma which is sometimes related with life or death of the newborn, curing using IFN-xcex12a known to have an antiangiogenic effect has been attempted (White C W., N. Engl. J. Med. 320, 1197-200, 1989) As remedies for these diseases, new antiangiogenic agents are desired eagerly.
There have been reports concerning several antiangiogenic substances so far. However, no effective substance which is fit for actual use has not been found yet (JP-A 3-109324, JP-A 3-236324 and JP-A 3-2148).
The present invention is to isolate a novel substance having an antiangiogenic effect to thereby provide an agent for preventing or treating various diseases accompanied by abnormal angiogenesis.
In view of the above situation, the inventors of the present invention have carried out research screening of antiangiogenic compound and as a result, found that an antiangiogenic compound is produced in a culture broth of microorganisms belonging to the genus Streptomyces. The active substance was isolated to determine the structure and as a result, it was clarified that this active substance was a novel active substance. Then the mechanism of action of the active substance was analyzed and as a result, found that this active substance had an inhibitory effect of the expression of adhesion molecule on endothelial cells as one of the mechanisms of inhibitory effects. Specifically, the substance showed a clear inhibitory effect on the expression of VCAM-1 and E-selectin of an endothelial cell. It is suggested that VCAM-1 and E-selectin are involved in the migration of endothelial cells and formation of lumens which are essential for angiogenesis (A. E. Koch et al, Nature, 376, 517, 1995). It is reported that the both are also involved in the adhesion of an endothelial cell to the lymph corpuscle. It is hence considered that the active substance is expected to be remedies for various diseases, specifically, autoimmune diseases, e.g., collagen diseases and graft rejection, cardiac infarction and arterial sclerosis, caused by an abnormal reaction of activated lymph corpuscles besides the diseases caused by an angiogenesis.
That is, the present invention relates to a chemical compound represented by the formula (1): 
(wherein, R1 represents hydrogen atom, aldehyde group or a lower acyl group, R2 and R3 are the same as or different from each other and represent hydrogen atom or a lower alkoxy group or are combined to represent an oxygen atom, R4 represents a lower alkyl group and R5 represents hydrogen atom or a lower alkyl group, provided that compounds in which R1 is aldehyde group, R2 and R3 are different from each other and are hydrogen atom or methoxy group, R4 is ethyl group and R5 is hydrogen atom are excluded), a salt therof or a hydrate thereof.
More specifically, it relates to a compound, a salt thereof or a hydrate thereof, in which, in the formula (1) R1 represents hydrogen atom, aldehyde group or acetyl group, R2 and R 3are the as same or different from each other and represent hydrogen atom or methoxy group or are combined to represent an oxygen atom, R4 represents ethyl group, normal propyl group or isopropyl group and R5 represents hydrogen atom or methyl group, provided that compounds in which R1 is aldehyde group, R2 and R3 which are different from each other and are hydrogen atom or methoxy group, R4 is ethyl group and R5 is hydrogen atom are excluded;
a compound, a salt thereof or a hydrate thereof, wherein, in the formula (1), R1 represents aldehyde group, R2 and R3 are the same as each other and represent hydrogen atoms, R 4 represents ethyl group and R5 represents hydrogen atom or methyl group;
a compound, a salt thereof or a hydrate thereof, wherein, in the formula (1), R1 represents aldehyde group, R2 and R3 are the same as each other and represent hydrogen atoms, R4 represents normal propyl group or isopropyl group and R5 represents hydrogen atom;
a compound, a salt thereof or a hydrate thereof, wherein, in the formula (1), R1 represents aldehyde group, R2and R3 are different from each other and represent hydrogen atom or methoxy group, R4 represents normal propyl group or isopropyl group and R5 represents hydrogen atom;
a compound, a salt thereof or a hydrate thereof, wherein, in the formula (1), R1 represents hydrogen atom, R2 and R3 are the same as each other and are combined to represent an oxygen atom, R4 represents ethyl group or isopropyl group and R5 represents hydrogen atom;
a compound, a salt thereof or a hydrate thereof, wherein, in the formula (1), R1 represents acetyl group, R2 and R3 are the same as each other and represent hydrogen atoms, R4 represents ethyl group and R5 represents hydrogen atom;
a medicament which comprises the above compounds or the compound in which, in the formula (1), R1 represents aldehyde group, R2 and R3 are different from each other and represent hydrogen atom or methoxy group, R4 represents ethyl group and R5 represents hydrogen atom, a salt thereof or a hydrate thereof as the active ingredient; and
a method of producing the above compounds or a salt thereof, which comprises culturing Streptomyces sp. Mer-VD1207, FERM P-15889 in a nutrient medium and collecting the above compounds or a salt thereof from the culture broth.
The present invention provides a medical composition which comprises a pharmacologically effective amount of the above compounds, a salt or a hydrate thereof and a pharmacologically acceptable carrier; a method for treating a disease against which an antiangiogenic effect or an effect of inhibiting the expression of an adhesion molecules VCAM-1 or/and E-selectin is efficacious, which comprises administering a pharmacologically effective amount of the above compounds, a salt thereof or a hydrate thereof to a patient; or use of the above compounds, a salt or a hydrate thereof for manufacturing a treating agent for a disease against which an antiangiogenic effect or an effect of inhibiting the expression of an adhesion molecules VCAM-1 or/and E-selectin is efficacious.
The present invention provides an agent for preventing or treating rheumatic arthritis, solid cancers, atherosclerosis, diabetic retinopathy, hemangioma and psoriasis.
As raw materials of the compound of the present invention, it is expected that any one of strains belonging to the genus Streptomyces can be used. However, as a typical strain used in the present invention, a strain which is actinomycetes separated from the soil collected in Kumamoto prefecture and was numbered as xe2x80x9cMer-VD1207 strainxe2x80x9d by the inventors is exemplified. The mycological properties of this Mer-VD1207 strain were as follows.
The cultivation was all carried out at 28xc2x0 C. The culture medium was selected from those recommended by International Streptomyces Project Committee (hereinafter referred to as ISP) The color tone is described by indicating the symbols shown in the parenthesis according to Color Harmony Manual of Container Corporation of America.
A microscopic inspection of the strain showed that vegetative mycelia elongated thoroughly and allowed aerial mycelia which likewise much elongated to adhere thereto. The apex of the aerial hyphae segmented into cylindrical spores. The external dimensions of the spore were 0.6-0.8 xcexcm-diameterxc3x970.8-1.0 xcexcm. The surface of the spore was smooth. The spores grew into a straight pore chain in which 10 to 50 pores were chained.
ISP-2 medium: The strain grew well and put forth reddish spores on poor aerial hyphae. The reverse side of colony was brown. The strain produced an yellowish brown-brown soluble pigment in the culture medium.
ISP-3 medium: The growth of the strain was not so well. And aerial mycelia were scant and white color. The reverse side colony was brown.
ISP-4 medium: The strain grew well and put forth reddish (5dc; Grayish yellowish pink) spores on rich aerial spores. The reverse side colony was yellowish brown.
ISP-5 medium: The strain grew moderately and put forth reddish spores on poor aerial hyphae. The reverse side colony was brown. The strain produced an yellowish brown-brown soluble pigment in the culture medium.
ISP-7 medium: The strain produced melanin pigment (the culture medium blacked).
The produced soluble pigments were not changed in color caused by pH.
Mycelium cake of the strain was inoculated on agar medium added each of various sugars ISP-9 and cultured for 3 to 10 days to observe its growth condition. The strain well-utilized glucose, fructose, rhamnose, sucrose, inositol, mannose, arabinose and xylose as carbon sources.
Mycelium cake of the strain was hydrolyzed using 6N HCl overnight. The concentrate was isolated using cellulose TLC and compared with a standard diaminopimelic acid to confirm its isomeric type. The isomeric type of the resulting diaminopimelic acid was LL type.
It is clear from the foregoing taxonomical study that the strain of the present invention belongs to the genus Streptomyces. The inventors of the present invention deposited this strain with National Institute of Bioscience and Human-Technology Agency of Industrial Science and Technology (Postal code 305-8566, 1-1-3, Higashi, Tsukuba-shi, Ibaraki-ken, Japan) as Streptomyces sp. Mer-VD1207 numbered as FERM P-15889 on Sep. 27, 1996. The strain was also transferred as international deposit No. FERM BP-6693 on Apr. 5, 1999.
Although a method of the cultivation of the aforementioned microorganisms conforms in principle to a method of the cultivation of ordinary microorganisms, preferably the cultivation is carried out in an aerobic condition such as those used in an aerobic stirring culture method in general. As the culture medium used in the cultivation, any culture medium containing a nutrient which can be utilized by microorganisms belonging to the genus Streptomyces may be used. Anyone of various synthetic media, semi-synthetic media, natural media and the like maybe used. As the composition of the medium, glucose, sucrose, fructose, glycerol, dextrin, starch, molasses, corn steep liquor, organic acids may be used as a carbon source singly or in combinations of two or more. As the nitrogen source, organic nitrogen source such as a pharmamedia, peptone, meat extract, yeast extract, soybean meal, casein, amino acid and urea and inorganic nitrogen source such as sodium nitrate and ammonium sulfate may be used singly or in combinations of two or more. A sodium salt, potassium salt, magnesium salt, phosphate and other salts of heavy metals may be added and used according to the need. It is to be noted that when conspicuous foaming is observed, various known defoaming agents may be added appropriately in the medium to the extent that the addition does not adversely affect the production of an object material.
The pH of the medium is preferably in an optimum pH range, generally, in the vicinity of a neutral zone. The temperature of cultivation is kept at generally 20 to 40xc2x0 C. and particularly preferably around 30xc2x0 C. at which the microorganisms grow well. Culture time is designed to be 1 to 5 days in the case of liquid culturing. The aforementioned various culture conditions maybe, of course, altered optionally corresponding to the types and qualities of the microorganisms to be used and external conditions. Also, corresponding to each of the aforementioned factors, optimum conditions are selected from the above range and adjusted.
In a method of screening the antiangiogenic effect, the degree of inhibition to the formation of lumens observed when an aortic fragment of a rat is cultured in a collagen gel was selected as an indicator (Nicosia R. F., Lab. Invest., 63, 115, 1990). Also, for the purpose of analyzing the mechanism of action of the antiangiogenic effect of the resulting compound, measurements were made concerning the effect of inhibiting the growth of endothelial cells by using endothelial cells derived from the human umbilical cord vein and the effect of inhibiting the expression of an adhesion molecules on endothelial cells.
The compound of the present invention can be purified from the culture broth of the microorganism as follows. The microorganism belonging the genus Streptomyces are cultured in a usual proper culture condition, the culture broth is then filtered and then extracted by an organic solvent such as butanol or methyl isobutyl ketone. The organic solvent layer is evaporated and then extracted using methanol, followed by treatment using light petroleum to obtain a crude extract. It is then fractionated by making proper use of adsorption chromatography using a silica gel or the like, LH 20 gel chromatography, partition chromatography, thin layer chromatography, paper chromatography or the like. An active fraction is confirmed by active screening. The foregoing measures are properly combined to isolate an active substance. As a solvent used in the adsorption chromatography, an organic solvent, which is usually used, such as chloroform, methanol, acetone, hexane or toluene is used. These solvents may be used by combining two or more while the concentration of the mixture solvent is appropriately selected. As a solvent used for crystallization, for example, chloroform and hexane or chloroform and carbon tetrachloride may be used. As one of the measures, there is a method developed by M. Lumb et al. (Nature, 206, 263, 1965). The structure of the isolated compound may be analyzed by measures according to a conventional method such as elemental analysis, GC-MS, NMR and melting point.
The inventors of the present invention have found that the isolated compound has a strong antiangiogenic effect. As aforementioned, based on the fact that abnormal arterialization is observed in various diseases, it is expected to serve as a preventive agent or a remedy for the diseases such as rheumatic arthritis, solid cancers, atherosclerosis, diabetic retinopathy, hemangioma and psoriasis and is effective especially as an anticancer agent.
When this compound is administered as a remedial or preventive agent for various diseases, it may be either orally administered in the form of a tablet, powder, granule, capsule or syrup, or parenterally administered as a spray, suppository, injection, external agent or drip agent. Although the dosage quite differs depending upon the degree of symptom, age and the type of hepatic disease, it is usually administered to an adult in an amount of about 1 mg to 100 mg for one to several portions a day.
In pharmaceutical preparation, a conventional pharmaceutical carrier is used to produce a target pharmaceutical by the usual method. That is, when a solid pharmaceutical for oral administration is prepared, a filler and, as required, further a binder, disintegrator, lubricant, colorant, flavoring agent are added to the base and then the resulting product is made into a tablet, coated tablet, granule, powder, capsule or the like by the usual method. These tablet and granule may be coated properly with, for example, a glaceing or gelatin coat according to the need without any problem.
When the injection is prepared, pH regulator, buffer, stabilizer, solubilizing agent and the like are added to the base if necessary, and the resulting product is made into an subcutaneous, intramuscular or intravenous injection by the usual method.